Method to detect fertilization potential of sperm

ABSTRACT

The invention relates to a method to detect male antifertility problems based on the determination of latent phospholipid hydroperoxide glutathione peroxidase (PHGPx).

This application is a National Stage Application filed under 371 fromInternational Application No. PCT/EP00/01877 filed Mar. 6, 2000, whichis a continuation of Application EP 00 103 959.5 filed Mar. 9, 1999.

FIELD OF THE INVENTION

The invention relates to a method to detect male antifertility problemsbased on the determination of latent phospholipid hydroperoxideglutathione peroxidase (PHGPx).

Selenium is essential for male fertility. In mature mammalianspermatozoa it is largely restricted to the midpiece harbouring thehelix of mitochondria embedded into a keratine-like selenium-enrichedmatrix called the mitochondrial capsule. Selenium deficiency isassociated with impaired sperm motility, structural alterations of themidpiece up to breakages, and loss of flagellum. The predominantselenoprotein of the mammalian male reproductive system, phospholipidhydroperoxide glutathione peroxidase (PHGPx), was shown to bepreferentially expressed in round spermatids but was hardly detectablein terms of messenger RNA or activity in spermatozoa. The basis of theinvention is the discovery that PHGPx persists in spermatozoa but asinsoluble, enzymatically inactive material forming the mitochondrialcapsule. PHGPx activity of this material can be restored by highconcentrations of thiols. PHGPx, thus, acts as a peroxidase in theproliferating germ epithelium to prevent oxidative damage. In the latestages of sperm maturation it is oxidatively cross-linked to become astructural element indispensible for sperm function. Accordingly, thedetermination of the PHGPx content in sperm or any other tissue ofhumans or livestock can be used to estimate the fertilization potentialof sperm.

SUMMARY OF THE INVENTION

The invention thus in accordance with claim 1 provides a method for thedetermination of latent phospholipid hydroperoxide glutathioneperoxidase (PHGPx) comprising the steps of

-   a) obtaining a sperm sample,-   b) solubilizing the spermatozoa by using detergents and chaotropic    agents and reactivating latent PHGPx by using high concentrations of    thiols and-   c) determining enzymatic activity of reactivated latent PHGPx.

In a further aspect the invention relates to the use of the inventivemethod in a method for predicting the fertilizing potential ofspermatozoa in sperm samples.

Further advantageous and/or preferred embodiments of the invention aresubject-matter of the subclaims.

In a preferred embodiment of the inventive method an additional step ofremoving any reactivating reagents by, e.g., gel filtration is providedbetween the step of solubilizing the spermatozoa and the step ofdetermining the enzymatic activity of reactivated latent PHGPx.

In a further embodiment of the invention instead of determiningenzymatic activity of reactivated latent PHGPx the content ofsolubilized PHGPx is determined by conventional immunological techniquesor measurement of enzymatic activity.

The used chaotropic a gent is, for example, 4-8 M guanidine chloride,4-8 M guanidine thiocyanate or 5-8 M urea.

The used thiol is, for example, 50-300 mM 2-mercaptoethanol, 25-300 mMdithiothreitol (DTT) or dithio-erythritol (DTE).

The sperm sample is, for example, from humans or livestock.

EXAMPLES

In the following the invention is disclosed in more detail withreference to examples and to drawings. However, the described specificforms or preferred embodiments are to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the followingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

Regarding the cited literature a reference list with more detailedbibliographic information can be found at the end of this specification.

Routine preparations of rat sperm mitochondrial capsules (1) yielded afraction which was insoluble in 1% SDS and 0.2 mM DTT and displayedexpected vesicular appearance in electron microscopy (FIG. 1 a). Thevesicles readily disintegrated upon exposure to 0.1 M mercaptoethanol(FIG. 1 b) and became fully soluble in 6 M guanidine-HCL. When thesolubilized capsule material was subjected to gel electrophoresisessentially four bands in the 20 kDa region were detected (FIG. 1 c,left lane). Western blotting revealed that the most prominent onereacted with antibodies directed against PHGPx (FIG. 1 c, right lane)which is undetectable as active peroxidase in mature spermatozoa (Tab.1). Also, N-terminal sequencing of the 21 kDa band representing about46% of total protein content according to Coomassie stain revealed thatit consisted of at least 95% pure PHGPx. Puzzled by this unexpected tofinding, we investigated the composition of the mitochondrial capsulesin more detail by 2D-electrophoresis (FIG. 2 a) followed bymicrosequencing and/or MALDI-TOF for identification (FIG. 2 b). For thispurpose the capsules were dissolved completely in a buffer designed forelectrophoretic separation of membrane proteins (see Methods). The spotmigrating with an apparent molecular weight of about 21 kDa andfocussing at a pH near 8 (spot 3) proved to be PHGPx according to themasses of tryptic peptides detected by MALDI-TOF (FIG. 2 b). By the sametechnique, also the slightly more acidic charge isomer (spot 4), themore basic ones (spots 1, 2 and 5) as well as the spots 6 and 7exhibiting a smaller apparent molecular mass were shown to contain PHGPx(FIG. 2 c). The predicted N-terminal (pos. 3-12) and C-terminal peptides(pos. 165-170), the fragment corresponding to positions 100-105 andthose expected from the basic sequence part 119-151 were too small to bereliably identified. Interestingly, the fragment corresponding topositions 34-48 comprising the active site selenocysteine was notdetected either. With these exceptions, however, the MALDI-TOF spectraunequivocally complied with the PHGPx sequence and thus proved thepresence of PHGPx in spots 1-7. On a thicker 2D-gel developed with anon-linear gradient from pH 3-10 also five distinct spots were detectedin the 20 kDa region. In this experiments the presence of PHGPx wasverified by microsequencing of major tryptic peptides (not shown). Againthe spots representing PHGPx were the most prominent ones present in thegel.

The spots 1-6 of FIG. 2 a proved to be essentially homogeneous. As isexemplified in FIG. 2 b, the fragments yielding MALDI-TOF signals ofsignificant intensities could be attributed to PHGPx. Only in the minorspot 7 a trace of impurity was detected, which was tentativelyidentified as a subunit of the T cell receptor variable region (acc. no.228109). Based on integrated stain intensities of the individual spotsthose representing PHGPx amounted to about 50% of the capsule material.Most of the minor components (see FIG. 2 a) are not likely constituentsof the capsule, which is believed to be built up by apposition ofextramitochondrial proteins onto the outer mitochondrial membrane. Inother gels further proteins like the mitochondrial glutathioneS-transferase subunit Yb-2 (acc. no. 121719) and an endothelinconverting enzyme (acc. no. 1706564) could be identified by MALDI-TOF ormicro-sequencing (not shown). Spots 8 and 9 were identified as the“outer dense fiber protein”, a cystine-rich structural sperm protein,which is associated with the helix of mitochondria in the sperm midpiecebut also extends into the flagellum (7). In view of the nature of theadditional proteins detected, the PHGPx content of the actualmitochondrial capsule should substantially exceed the 50% observed bygel scanning.

Despite intense search, we could not detect any trace of the “spermmitochondria-associated cysteine-rich protein (“SMCP”) (7) in ourcapsule preparation. This cysteine- and proline-rich protein had forlong been considered the selenoprotein accounting for the seleniumcontent of the mitochondrial capsule in sperm (1,8,9). Cloning of therat SMCP gene, however, revealed that it did not contain any in-frameTGA codon enabling selenocysteine incorporation (10). In mice, the threein-frame TGA codons proved to be upstream of the translation start (7).In developing mouse sperm SMCP stayed cytosolic up to states in whichthe mitochondrial capsule was already formed and only becamesuperficially associated with the outer mitochondrial membranes of latespermatids and epididymal spermatozoa (7). SMCP thus is not necessarilyan integral part of the mitochondrial capsule nor it is a selenoprotein.Instead, the “mitochondrial capsule selenoprotein (MCS)”, as SMCP wasoriginally referred to (1,7-10), is indeed PHGPx.

The chemical modifications of PHGPx leading to distinct differences incharge and apparent MW could not be reliably elucidated. Sequencingrevealed an identical N-terminus of the size isomers starting withASRDDWRCAR, i.e. a sequence either corresponging to the originallyproposed translation start (11) after cleavage of the first two residuesor derived from a possible pre-PHGPx (12) after processing of amitochondrial leader peptide (13). Tryptic fragments extending towardsthe C-terminus up to position 164 were consistently observed also withthe faster migrating specimen (FIG. 2 c) which leaves little room toexplain an apparent MW difference of 1 to 1.5 kDa. As to the chargeisomers, it may be recalled that a potential phophorylation had beeninferred from early attempts to sequence pig heart PHGPx (14). Theassignment of masses to possibly phosphorylated tryptic peptides,however, remained equivocal. Certainly, more trivial events such asdeaminations of Gln and Asn residues, C-terminal degradation, oxidationof the active site selenium, or its elimination might have contributedto the charge heterogeneity.

DETAILED DESCRIPTION OF THE INVENTION

PHGPx as the major component of the sperm mitochondrial capsule had sofar escaped attention, since as such it is enzymatically inactive, as itgenerally is in mature spermatozoa prepared from the tail of theepididymis (Tab. 1). It is neither reactivated by glutathione in the lowmillimolar range as used under conventional test conditions. Highconcentrations of thiols (0.1 M 2-mercaptoethanol or dithiothreitol),which in the presence of guanidine fully dissolve the capsule,regenerate a significant PHGPx activity, as measured after eliminationof denaturating and reducing agents (Tab. 1). In fact, the specificactivities thus obtained from mitochondrial capsules exceed, by a factorof 20, the highest values ever measured, i.e. in spermatogenic cells.Nevertheless, this extreme PHGPx activity is still low compared to itscontent in PHGPx protein. Based on the specific activity of pure PHGPx,the reactivated enzyme would be equivalent to less than 3% of thecapsule protein, whereas the 2D-electrophoresis suggests a PHGPx proteincontent of at least 50%. It is worth noting that the same reductiveprocedure does not increase the specific activity of PHGPx inspermatogenic cells from testicular tubules (Tab. 1). The switch ofPHGPx from a soluble active enzyme to an enzymatically inactivestructural protein thus occurs during final differentiation ofspermatozoa.

The alternate roles of PHGPx, being either a glutathione-dependenthydroperoxide reductase or a structural protein, are not necessarilyunrelated. One of the features common to all glutathione peroxidases isa selenocysteine residue which together with a tryptophan and aglutamine residue forms a catalytic triad (15,16). Therein the selenolgroup of the selenocysteine residue is dissociated and highly activatedby hydrogen bonding to reduce hydroperoxides with high rate constants.The reaction product, a selenenic acid derivative, R-SeOH, will readilyreact with thiols, e.g. GSH, to form an intermediate with aselenadisulfide bridge between enzyme and substrate, R-Se-S-G, fromwhich the ground state enzyme can be regenerated by a second GSH. PHGPxis unique among the glutathione peroxidases in several respects: i) Itusually is monomeric having its active site freely accessible at thesurface; this facilitates interaction with bulky substrates. ii)Arginine residues surrounding the active site and specifically bindingglutathione in most types of glutathione peroxidases are lacking inPHGPx (16); correspondingly, its specificity for the reducing substrateis less pronounced (16). It therefore can be envisaged that oxidizedPHGPx may form diselenide or selenadisulfide bridges with exposed SeH orSH groups of proteins (16) including PHGPx itself, and this process,possibly followed by SE/SS, SH/SeS, or SH/SeSe exchange reactions, willcreate cross-linked protein aggregates. This ability of PHGPx mightbecome particularly important if cells are exposed to hydroperoxides atextremely low concentration of glutathione, as is documented for latestates of spermatogenesis (17-20). FIG. 3 is to mimick the oxidativeevents occurring during sperm maturation. Short term exposure of solubleproteins derived from spermatogenic cells to moderate H₂O₂concentrations in the absence of GSH yields a variety ofPHGPx-containing high molecular weight aggregates. Undoubtedly,therefore, PHGPx, by means of its intrinsic enzymatic potential, cancatalyse oxidative protein aggregation using protein thiols as alternatesubstrates. During sperm maturation, PHGPx thereby transforms itselfinto an enzymatically inactivated structural protein. This view,however, is not to imply that PHGPx could not depend on additionalproteins when building up the highly organized architecture of thespermatozoal midpiece.

Our findings require a fundamental reconsideration of the role ofselenium in male fertilty. The intriguing predominance of theselenoprotein PHGPx in the male reproductive system has so far beenbelieved to reflect the necessity to shield germ line cells fromoxidative damage by hydroperoxides and reactive oxygen species derivedtherefrom (11,17,21,22). This concept still merits attention with regardto the mutagenic potential of hydroperoxides and probably holds true forthe early phases of spermatogenesis where PHGPx is still present as anactive peroxidase (6,21). At this stage related activities reported forPHGPx or other glutathione peroxidases, e.g. silencing lipoxygenases(23), dampening the activation of NF B (24) or inhibiting apoptosis(25), may also be relevant. In later stages of spermatogenesischaracterized by a shift of the redox status resulting in loss of GSH(18-20,26), the ability of PHGPx to use protein thiols as alternatesubstrates opens up new perspectives of redox regulation which remain tobe explored. In the mature spermatozoon PHGPx has experienced apronounced metamorphosis now being a major constituent of thekeratinuous material embedding the mitochondrial helix. It appearsrevealing that precisely this architectural pecularity in the midpieceof spermatozoa shows gross structural alterations in seleniumdeficiency. We therefore assume that the mechanical instability of themidpiece observed in selenium deficiency is a consequence of an impairedPHGPx biosynthesis. This view implies that it is not the antioxidantcapacity of PHGPx which is crucial for male fertility but its ability toutilize hydroperoxides to build an indispensable structural element ofthe spermatozoon.

Any shortage of PHGPx during sperm maturation, be it due to seleniumdeficiency, other reasons of inhibited biosynthesis or inhibition ofactivity should therefore result in disturbed sperm midpiecearchitecture and, in consequence, loss of fertilization potential ofsperm. This conclusion was further corroborated by determination ofreactivated PHGPx in sperm of individuals with documented fertilityproblems. The latter were divided into three groups: depending onwhether i) intrauterine sperm injection (iui) or ii) conventionalin-vitro-fertilisation (ivt-et) was still successful or iii)intracytoplasmatic sperm injection was required (icsi). As shown in FIG.4, the PHGPx values differed markedly between these groups. While theiui group displayed values close to normal, PHGPx in the icsi group wasalmost absent, the ivf-et group ranking in between. The reasons of thediverse PHGPx content being unknown, the data reveal that markedlyreduced PHGPx content in sperm is incompatible with normal malefertility. Similarly, there is a strong correlation between “typical”sperm appearance (FIG. 5) and “fast” moving sperm with PHGPx content(FIG. 6). This correlation, however, shows marked scattering of dataindicating that PHGPx content of sperm is not the only reason ofabnormal shape and motillity of sperm. It should also be pointed outthat the sperm samples were taken from individuals without any obviousdisease suggesting that extremely reduced PHGPx levels are welltolerated.

Taken together, the ovservation that PHGPx builds up an esssentialstructure of sperm and that its content in sperm correlates with thefertilization potential leads to the inventive concept to use the PHGPxcontent of sperm as a predictive parameter for the necessary measures toovercome male fertility problems. To this end, it appears necessary toreactivate the PHGPx contained in sperm in order to estimate its contentby either immunological methods or by any of the establisheddeterminations of its specific activity. (28, 29).

Methods

Preparation of Rat Spermatozoa, Tubular Cells and Mitochondrial Capsule

Spermatozoa of four month old Wistar rats (about 300 grams of bodyweight) were collected by squeezing cauda epididymis and vas deferens inphosphate buffer saline (PBS) and by centrifugating at 600×g for 10minutes. Cell and sperm pellets were layered on a discontinous 45%, 70%and 95% Percoll gradient and centrifugated at 300×g for 20 min.Spermatogenic cells stacked on top of the gradient, while spermatozoaseparated into the 70% Percoll layer. Cells from seminiferous epitheliumwere prepared as follows (26): testes were deprived of albuginea,seminiferous tubules were cut into small pieces in PBS containing 0.250mg/ml collagenase, and incubated twice 25° C. for 15 min. Cells thenwere filtered through a stainless steel screen (140 μm pore), washed inPBS and centrifugated at 300×g for 10 min. Sperm mitochondrial capsulewas prepared according to Calvin et al. (1): sperms were resuspended in0.05 M Tris-HCl pH 8.0 at the concentration of 10⁶ cells/ml and treatedwith trypsin (0.2 mg/ml) for 10 minutes. After stopping the proteaseaction with trypsin inhibitor (0.5 mg/ml) and SDS (10 mg/ml) sperms werecentrifugated at 1,500×g for 10 minutes. Pellets were resuspended in0.05 M Tris-HCl, pH 8.5 containing 1% sodium dodecyl sulphate (SDS), and0.2 mM DTT and kept under continuous stirring for 30 minutes. Followingcentrifugation at 4,500×g for 15 min, the resulting supernatant waslayered on a 1.6 M sucrose cushion. After centrifugation for 20 min at18,000×g in a swinging rotor, sperm capsules were collected as a band atthe top of the sucrose cushion, washed in Tris-HCl, pH 8.0 and spun at140,000×g.

1D-Electrophoresis and Western Blotting

Electrophoresis was performed according to Laemmli under either reducing(+2−mercaptoethanol) or non-reducing conditions. Proteins were blottedonto nitrocellulose, probed with an antigen-purified rabbit antibodyraised against pig heart PHGPx and detected by biotinylated anti rabbitIgG and streptoavidin alkaline phosphatase complex.

2D-Electrophoresis

100 μg of the mitochondrial capsule material was dissolved in 400 μl ofa solution containing of 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, 20mM Tris base and 0.5% IPG buffer (Pharmacia) and focused in an IPG-phor(Pharmacia) 2-D gel electrophoresis system at 20° C. by stepwiseincreasing voltage up to 5000 V but not exceeding a current of 30 μA perIPG strip. The pH gradient was non-linear from 3-10 or linear from 3-10or 6-11. The focused IPG strips were then equilibrated for SDSelectrophoresis (10 min each) with a solution containing 60 mM DTT in 6M urea, 30% glycerol, 0.05 M Tris-HCl buffer pH 8.8 and in the samebuffer where DTT was substituted by 250 mM iodoacetamide. AfterSDS-electrophoresis (12% polyacrylamide) the gels were stained withCoomassie.

Protein Identification

Coomassie-stained spots were cut out from the gels, neutralized with(NH₄)HCO₃, destained with 400 μl 50% acetonitrile/10 mM (NH₄)HCO₃ anddried in a Speed Vac Concentrator. Protein digestion was done overnightusing 2 ng/μl sequencing grade trypsin (Promega) in 50 mM (NH₄)HCO₃(Boehringer, Mannheim). The resulting peptides were extracted twice with60% acetonitrile/40% H₂O/0.1% TFA. Extracts were combined andlyophilized in the Speed Vac Concentrator. Peptide digests were desaltedon small RP18-columns, eluted with saturated α-hydroxy-4-cyano-cinnamicacid and loaded directly onto the MALDI target (27). Reflectron MALDImass spectra were recorded on a Reflex™ MALDI/TOF-mass spectrometer(Bruker-Franzen-Analytik, Bremen). The ions were excellerated at 20 kVand reflected at 21.3 kV. Spectra were externally calibrated using themonoisotopic MH⁺ion from two peptide standards. 100-200 laser shots weresummed up for a single mass spectrum. Mass identification was performedwith MS-Fit (http://falcon.ludwig.ucl.ac.uk/ucsfhtml/msfit.htm).

Alternatively, protein spots from 1.5 mm 2D-gels were digested withmodified trypsin (Promega, sequencing grade) in 25 mM (NH₄)HCO₃overnight at 37° C. The digests were extracted twice and dried as beforeand reconstituted in 10 μl water. Peptides were separated on areversed-phase capillary column (0.5 mm×150 mm) with a gradient ofacetonitrile in 0.1% formic acid/4 mM ammonium acetate at a flow rate of5 μl/min and collected manually. Aliquots of 5 μl were spotted ontoBiobrene-treated glass fiber filters and sequenced on an AppliedBiosystems 494A sequencer with standard pulsed-liquid cycles. BeforeN-terminal sequencing, proteins were blotted from polyacrylamide gelsonto PVDF membranes for 16 h at pH 8.3 (25 mM Tris-HCl, 192 mM glycine)and 100 mA (30 V).

When applicable, PHGPx was also identified by activity measurementaccording to (28) using the specific substrate phosphatidylcholinehydroperoxide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Presence of PHGPx in the mitochondrial capsule of spermatozoa.

a, Mitochondrial capsule prepared by trypsination and centrifugationaccording to (1) at 80,000 fold magnification.

b, The same preparation as shown in a, but after exposure to 0.1 M2-mercaptoethanol for 15 min at 4° C. Contamination of the capsulematerial by mitochondria is evident from the presence of mitochondrialghosts. c, SDS gel electrophoresis of proteins extracted from capsulematerial (see Methods) by treatment with 0.1 M 2-mercaptoethanol, 0.1 MTris-HCl, pH 7.5, and 8 M guanidine HCl. Left lane is stained withCoomassie, right lane demonstrates presence of PHGPx by Westernblotting.

FIG. 2 showws the analysis of the composition of the mitochondrialcapsule of spermatozoa

a, 2D-electrophoresis of purified dissolved capsule material. Proteinswere focused in a linear pH-gradient from 3 to 10 (horizontaldirection), then reduced, amidocarboxymethylated, subjected toSDS-electrophoresis, and stained with Coomassie. MALDI-TOF analysis ofthe visible spots identified the following proteins (SwissProt database): spot 1-7 PHGPx (MW 19 443; pI 8.27; acc. no. 544434); spots 8 and9, outer dense fiber protein (MW 27351; pI 8.36; acc. no. P21769); spots10 and 11, voltage-dependent anion channel-like protein (MW 31720; pI7.44; acc. no. 540011); spot 12, “stress-activated protein kinase” (MW48107; pI 5.65; acc. no. 493207); spot 13, glycerol-3-phosphatedehydrogenase (MW 76479; pI 5.86; acc. no. P35571).

b, MALDI-TOF spectrum (overview) of tryptic peptides obtained from PHGPxas found in spot 3. Abscissa, mass/charge ratio of the peptidefragments; ordinate, arbitrary units of intensity; numbers at masssignals, identified peptides in the PHGPx sequence (see insert forposition numbers); T, trypsin-derived fragments.

c, Compilation of tryptic PHGPx fragments identified in spots 1-7 byMALDI-TOF. Vertical lines designate potential tryptic lag cleavagesites. Dark blocks, identified typical cleavage products; shadowedblocks, masses resulting from incomplete cleavage or equivocallyassignable to different fragments (e.g. 3-9 and 63-69).

FIG. 3 shows the formation of PHGPx-containing aggregates fromspermatogenetic cells by H₂O₂ in the absence of GSH. Spermatogenic cellswere homogenised in 0.1 M Tris-HCl, 6 M guanidine-HCl, 0.5 μg/mlpepstatin A, 0.7 μg/ml leupeptin and 5 mM 2-mercaptoethanol at pH 7.5and 4° C. After centrifugation at 105,000×g for 30 min, excess reagentswere removed by gel permeation using NAP 5 columns equilibrated with 10mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA and 0.1% Triton X-100, pH 7.5.Immediately (t 0) and 15 min after (t 15) the addition of 75 μM H₂O₂aliquots of the mixture (0.05 mg of protein) were withdrawn andsubjected to electrophoresis under (a) reducing and (b) non reducingconditions. After blotting on nitrocellulose, PHGPx was detected byspecific antibodies.

FIG. 4 shows the PHGPx specific activity in extracts (0.1% Triton X-100and 0.1 M 2-mercaptoethanol of human sperm. Correlation between thisparameter and therapeutic appproach in cases of couple infertility.

FIG. 5 shows the relationship between PHGPx specific activity and numberof “typical” sperms per milliliter of semen. “Typical” is amorphological parameter of sperm evaluation.

FIG. 6 shows the relationship between PHGPx specific activity and numberof “fast” sperms per milliliter of semen. “Fast” is a parameter of spermmobility.

TABLE 1 PHGPx activity in spermatogenic cells, spermatozoa and spermcapsule. Effect of thiols. Preparation mU/mg protein^(a,b) Cells fromseminiferous tubules  5 mM 2-mercaptoethanol^(c) 250 ± 10 100 mM2-mercaptoethanol^(c) 260 ± 10 Spermatozoa from tail of epididymis  5 mM2-mercaptoethanol^(c) undetectable 100 mM 2-mercaptoethanol^(c) 3,140 ±200  Mitochondrial capsule  5 mM 2-mercaptoethanol^(c) undetectable 100mM 2-mercaptoethanol^(c) 5,600 ± 290  ^(a)One enzyme mU catalyzes thereduction of one nanomole of phosphatidylcholine hydroperoxide perminute at 37° C. in the presence of 3 mM GSH. ^(b)Data are the mean andS. D. of five independent measurements. ^(c)Solubilisation/reduction wascarried out in 0.1M Tris-HCl, 6M guanidine-HCl, 0.5 μg/ml pepstatin A,0.7 μg/ml leupeptin and 2-mercaptoethanol as indicated at pH 7.5 and 4°C. for 10 min Low molecular weight compounds were removed beforeactivity determination by a NAP 5 cartridge.

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1. A method for determining the concentration of latent phospholipidhydroperoxide glutathione peroxidase (PHGPx) in a sperm sample,comprising the steps of: a. obtaining the sperm sample, b. solubilizingspermatozoa in said sperm sample by adding detergents and chaotropicagents; c. reactivating latent PHGPx by adding thiols; d. removing thechaotropic agents and thiols from the sample; and, e. determining theconcentration of the latent PHGPx.
 2. The method according to claim 1,wherein the chaotropic agents and thiols are removed by gel filtration.3. The method according to claim 1, wherein the concentration ofsolubilized PHGPx is determined by immunological techniques ormeasurement of enzymatic activity of said solubilized PHGPx.
 4. Themethod according to claim 1, wherein the chaotropic agent is 4-8 Mguanidine chloride, 4-8 M guanidine thiocyanate or 5-8 M urea.
 5. Themethod according to claim 1, wherein the thiol is 50-300 mM2-mercaptoethanol, 25-300 mM dithiothreitol (DTT) or dithioerythritol(DTE).
 6. The method according to claim 1, wherein the sperm sample isfrom humans or livestock.
 7. The method according to claim 1, comprisingthe step of calculating fertilizing potential of said spermatozoa byusing the concentration of latent PHGPx.